In modern molecular biology and genetic engineering, many molecular techniques that involve the use of molecules of nucleic acid often require the generation of a supply of nucleic acid molecules by synthetic methods. For example, to test hypotheses in the field of metabolic engineering or genomics, and to synthesize designed proteins and organisms with tailored genomes, cost-effective methods for synthesizing nucleic acid molecules with a high degree of fidelity to an intended nucleotide sequence are often required. Common methods of nucleic acid synthesis, e.g. synthesis of double-stranded DNA, include polymerase chain reaction methods and ligation chain reaction methods. Often, ensuring that a synthetic DNA molecule contains the correct nucleotide sequence is important, if not essential, for the success of the molecular technique in which the synthesized DNA is to be used. For example, the synthesis of a DNA coding sequence for use in gene expression of functional polypeptides requires a precise DNA sequence; because even one nucleotide substitution, insertion or deletion can have significant consequences for the polypeptide that is ultimately produced. Thus, the process of minimizing DNA molecules having incorrect DNA sequences from a synthetic DNA population is widely considered to be essential in providing error-free synthetic DNA produced by a de novo gene synthesis method.
Recently, efforts to synthesize nucleic acid molecules accurately while controlling costs have yielded methods including microchip-based gene synthesis and PCR-based gene assembly technologies. While these conventional technologies provide the capability to synthesize multiple genes, reducing errors introduced into the desired gene-sequence remains challenging. To avoid the problems with sequence errors inherent in gene synthesis, some have focused on purifying the oligonucleotides that are used at the early stages of the synthesis process. However, these oligonucleotide purification approaches are costly, and sequence errors persist and propagate through the subsequent steps of the synthesis process.
Thus, there exists a need for alternative methods for reducing sequence errors within a population of DNA molecules. What is desired is a way to synthesize genes and other nucleic acid molecules with a greater yield of molecules having a desired nucleotide sequence. An approach that can correct sequence errors at a much later step in the synthesis process makes the desired increase in nucleotide sequence accuracy possible, while allowing the process to be cost-effective.